Biomedical subjects

S B Liggett

Publications and source records attributed to S B Liggett.

At least 91 records · Page 5Linked to original sources

PubMed1995

Genetic polymorphisms of the beta 2-adrenergic receptor in nocturnal and nonnocturnal asthma. Evidence that Gly16 correlates with the nocturnal phenotype.

Nocturnal asthma represents a unique subset of patients with asthma who experience worsening symptoms and airflow obstruction at night. The basis for this phenotype of asthma is not known, but beta 2-adrenergic receptors (beta 2AR) are known to downregulate overnight in nocturnal asthmatics but not normal subjects or nonnocturnal asthmatics. We have recently delineated three polymorphic loci within the coding block of the beta 2AR which alter amino acids at positions 16, 27, and 164 and impart specific biochemical and pharmacologic phenotypes to the receptor. In site-directed mutagenesis/recombinant expression studies we have found that glycine at position 16 (Gly16) imparts an accelerated agonist-promoted downregulation of beta 2AR as compared to arginine at this position (Arg16). We hypothesized that Gly16 might be overrepresented in nocturnal asthmatics and thus determined the beta 2AR genotypes of two well-defined asthmatic cohorts: 23 nocturnal asthmatics with 34 +/- 2% nocturnal depression of peak expiratory flow rates, and 22 nonnocturnal asthmatics with virtually no such depression (2.3 +/- 0.8%). The frequency of the Gly16 allele was 80.4% in the nocturnal group as compared to 52.2% in the nonnocturnal group, while the Arg16 allele was present in 19.6 and 47.8%, respectively. This overrepresentation of the Gly16 allele in nocturnal asthma was significant at P = 0.007 with an odds ratio of having nocturnal asthma and the Gly16 polymorphism being 3.8. Comparisons of the two cohorts as to homozygosity for Gly16, homozygosity for Arg16, or heterozygosity were also consistent with segregation of Gly16 with nocturnal asthma. There was no difference in the frequency of polymorphisms at loci 27 (Gln27 or Glu27) and 164 (Thr164 or Ile164) between the two groups. Thus the Gly16 polymorphism of the beta 2AR, which imparts an enhanced downregulation of receptor number, is overrepresented in nocturnal asthma and appears to be an important genetic factor in the expression of this asthmatic phenotype.

Alleles↗

PubMed1994

The palmitoylated cysteine of the cytoplasmic tail of alpha 2A-adrenergic receptors confers subtype-specific agonist-promoted downregulation.

Most guanine nucleotide binding protein (G protein)-coupled receptors have a conserved cysteine in the C-terminal cytoplasmic tail near the seventh transmembrane spanning region. This cysteine is known to be palmitoylated in rhodopsin, the beta 2-adrenergic receptor (beta 2AR) and the alpha 2A-adrenergic receptor (alpha 2AAR). For the beta 2AR, this cysteine has been shown to be important for stimulatory G protein (Gs) coupling and agonist-promoted desensitization. For the alpha 2AAR (human alpha 2 C10) palmitoylation occurs at Cys-442, but it is not known what function such fatty acid acylation subserves. The closely related alpha 2CAR subtype denoted alpha 2C4 lacks a cysteine in this region and has different G-protein-coupling characteristics and agonist regulatory properties as compared to alpha 2C10. To assess the role of the palmitoylcysteine in alpha 2AR function, we constructed a mutated alpha 2C10 having a phenylalanine (the analogous amino acid in the alpha 2C4 in this position) substituted for Cys-442, denoted alpha 2C10(Phe-442), and expressed this along with wild-type alpha 2C10 and alpha 2C4 in CHO cells. Functional coupling to inhibitory G protein (Gi) and to Gs was identical between wild-type alpha 2C10 and alpha 2C10(Phe-442). Agonist-promoted desensitization of both the Gi and Gs-mediated pathways was also found to be unaffected by this mutation. Cellular trafficking induced by agonist exposure was evaluated by delineation of intracellular (sequestered) versus cell surface receptors and by determination of net receptor loss. Mutation of Cys-442 did not alter the extent or rate of agonist-promoted sequestration induced by agonists or the recovery from sequestration. However, the downregulation of receptor number after prolonged agonist exposure was completely abolished by this mutation and converted alpha 2C10 to an alpha 2C4 phenotype in regard to this adaptive response. Another mutated alpha 2C10, in which Cys-442 was replaced by alanine, also failed to downregulate. Thus, the function of this cytoplasmic palmitoylcysteine is distinctly different between the alpha 2AR and other G-protein-coupled receptors such as the beta 2AR and rhodopsin, and this suggests that this molecular attribute may subserve diverse roles among members of this family of receptors. For the alpha 2ARs, this may represent an evolved feature that provides for differing needs for regulation of the alpha 2C10 and alpha 2C4 subtypes by agonist.

Adenylyl Cyclases↗

PubMed1994

A proline-rich region of the third intracellular loop imparts phenotypic beta 1-versus beta 2-adrenergic receptor coupling and sequestration.

beta-Adrenergic receptor (beta AR) subtypes differ not only by characteristic ligand affinities but also in the manner and extent to which they mediate agonist-promoted events such as activation of adenylyl cyclase and receptor sequestration. We utilized mutagenesis and recombinant expression in Chinese hamster fibroblasts to examine the effect of an unusual proline-rich 24-amino acid sequence (PARPPSPSPSPVPAPAPPPGPPRP) present in the third intracellular loop of the beta 1AR, but not in the beta 2AR, on the aforementioned receptor-mediated events. Cells expressing the wild-type beta 2AR stimulated adenylyl cyclase in response to the agonist isoproterenol with an EC50 approximately 5-fold lower than that observed with the beta 1AR (0.53 +/- 0.14 versus 2.47 +/- 0.52 nM, p < 0.01). Deletion of the proline-rich sequence from the beta 1AR resulted in an improvement in isoproterenol-stimulated adenylyl cyclase to an EC50 value intermediate to that observed in the wild-type receptors (1.14 +/- 0.08 nM, p < 0.05 versus wild-type beta 1AR). In contrast, insertion of this sequence into the beta 2AR impaired its ability to mediate this process. Similar results were observed for receptor sequestration. Wild-type beta 1-and beta 2AR underwent maximal agonist-promoted sequestration of 25.9 +/- 4.0 and 60.0 +/- 3.3%, respectively. Deletion of the proline-rich region from the beta 1AR improved maximal sequestration to 43.0 +/- 2.7% (p < 0.01 versus wild-type beta 1AR), while insertion of the sequence into the beta 2AR impaired sequestration to 33.9 +/- 2.7% (p < 0.001 versus wild-type beta 2AR). We conclude that the distinct phenotypic patterns observed for these two agonist-promoted events in the beta 1AR and beta 2AR subtypes are partially due to the conformational effects of this proline-rich third intracellular loop sequence. Such regions, which are also found in some other G-protein-coupled receptors, may represent a general motif responsible for attenuating certain agonist-promoted receptor events.

Adenylyl Cyclases↗

PubMed1994

Amino-terminal polymorphisms of the human beta 2-adrenergic receptor impart distinct agonist-promoted regulatory properties.

We have recently delineated three naturally occurring polymorphisms of the human beta 2-adrenergic receptor caused by missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the receptor. We have studied the functional consequences of these polymorphisms by site-directed mutagenesis and the recombinant expression of these receptors in Chinese hamster fibroblasts. The polymorphisms consist of substitutions of Gly for Arg at amino acid 16 (Arg16-->Gly), Glu for Gln at amino acid 27 (Gln27-->Glu), and a combination of both substitutions. All three mutated receptors displayed normal agonist binding and functional coupling to Gs, resulting in the stimulation of adenylyl cyclase activity. However, these mutations markedly altered the degree of agonist-promoted downregulation of receptor expression. After 24-h exposure to 10 microM isoproterenol, wild-type beta 2AR underwent a 26 +/- 3% reduction in receptor density. In contrast, Arg16-->Gly underwent a 41 +/- 3% reduction. Gln27-->Glu, on the other hand, was found to be completely resistant to downregulation. Arg16-->Gly+Gln27-->Glu also underwent an increased downregulation compared to wild-type beta 2AR (39 +/- 4%). The rates of new receptor synthesis after irreversible alkylation were not different between these receptors, nor were the rates of agonist-promoted receptor internalization to the intracellular pool. Gln27-->Glu cellular mRNA minimally increased during agonist exposure, and wild-type beta 2AR and the other mutated receptor mRNAs did not change, which infer that the aberrant downregulation patterns of these polymorphisms may be due to the altered degradation of receptor protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1994

Contribution of ligand structure to activation of alpha 2-adrenergic receptor subtype coupling to Gs.

Recently, we have demonstrated that alpha 2-adrenergic receptors (alpha 2AR) functionally couple not only to Gi but also to Gs. This alpha 2AR-Gs coupling was subtype selective, in that the degree of alpha 2AR-Gs (but not -Gi) coupling was different between alpha 2AR subtypes. It is not known whether the determinants of this subtype selectively are found within the ligand-binding region of the receptor or within the intracellular G protein-coupling domains of the individual subtypes. We therefore expressed the three cloned human alpha 2AR (alpha 2C10, alpha 2C4, and alpha 2C2) in Chinese hamster ovary cells and studied the contribution of the ligand-binding domain to functional Gi versus Gs coupling, by determining the ability of various agonists (catecholamines, imidazolines, and azepines) to elicit alpha 2AR-mediated inhibition and stimulation of adenylyl cyclase activity. Isolation of Gi and Gs responses was accomplished by incubating cells with cholera or pertussis toxin, respectively. Although each compound was found to be a full agonist for alpha 2AR-Gi coupling, the efficacy of these agonists to elicit alpha 2AR-Gs coupling was markedly different, not only among drugs but also among the three alpha 2AR subtypes. The most notable differences occurred with the imidazoline agonists. Specifically, oxymetazoline stimulated adenylyl cyclase activity 210 +/- 17% for alpha 2C2 and 22 +/- 2.6% for alpha 2C10 and displayed no stimulation for alpha 2C4. UK-14304 stimulated adenylyl cyclase activity 240 +/- 16% for alpha 2C10, 160 +/- 14% for alpha 2C4, and 86 +/- 9% for alpha 2C2. Overall, the rank order of efficacy of these agonists to elicit stimulation of adenylyl cyclase activity by alpha 2C10 was epinephrine = norepinephrine = UK-14304 > BHT-933 > BHT-920 > oxymetazoline. For alpha 2C4 the rank was epinephrine = norepinephrine = UK-14304, with oxymetazoline, BHT-920, and BHT-933 not eliciting any stimulation. For alpha 2C2 the rank was epinephrine = norepinephrine > oxymetazoline > UK-14304 = BHT-920 > BHT-933. Thus, the coupling of alpha 2AR subtypes to Gs occurs with endogenous catecholamines as well as multiple synthetic agonists, and the degree of Gs coupling is highly dependent on the structure of the agonist. Also, compounds that act as full agonists for Gi coupling are not necessarily full agonists for Gs coupling.

Adenylyl Cyclases↗

PubMed1993

A polymorphism of the human beta 2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor.

We have recently identified several naturally occurring variants of the human beta 2-adrenergic receptor (beta 2AR). One of these polymorphisms, which is relatively uncommon, is a mutation occurring in the fourth transmembrane spanning domain, with Ile substituted for Thr at amino acid 164 within the proposed ligand binding pocket. This mutation is adjacent to Ser165 which has been predicted to interact with the beta-carbon hydroxyl group of adrenergic ligands. To determine the functional significance of this variant, we constructed by site-directed techniques a mutated beta 2AR (Ile164) with this substitution and expressed it in CHW-1102 cells. In the presence of guanine nucleotide, Ile164 displayed a lower binding affinity for epinephrine as compared with the wild-type beta 2AR (Ki = 1450 +/- 79 versus 368 +/- 39 nM; p < 0.001). A similarly decreased affinity was found with the catecholamines isoproterenol and norepinephrine, but not with dobutamine or dopamine which lack hydroxyl groups on their beta-carbons. In addition, antagonists without aromatic ring polar substituents displayed a decreased affinity for the mutated receptor. In agonist competition experiments conducted in the absence of guanine nucleotide, Ile164 failed to exhibit detectable high affinity binding, suggesting an impairment in the formation of the agonist-receptor-Gs complex. Consistent with this finding, functional coupling to Gs as determined in adenylyl cyclase assays was significantly (approximately 50%) depressed with Ile164 under both basal and agonist-stimulated conditions. beta 2AR sequestration, which is also triggered by agonist binding, was also found to be approximately 65% reduced in the Ile164 polymorphism. This study represents the first characterization of a naturally occurring mutation of a human adrenergic receptor. Our findings generally support the hypothesized role of this region of the receptor for ligand binding and receptor activation, as well as for establishing critical interactions for overall receptor conformation.

Adenylyl Cyclases↗

PubMed1993

Functional alpha 2-adrenergic receptor-Gs coupling undergoes agonist-promoted desensitization in a subtype-selective manner.

Recently it has become clear that alpha 2-adrenergic receptors (alpha 2AR) functionally couple to Gs as well as Gi, thus inducing a complex modulation of adenylyl cyclase activity. It is unknown whether alpha 2AR-Gs coupling undergoes agonist-promoted desensitization. Therefore, in CHO cells expressing the three cloned human alpha 2AR subtypes (alpha 2C10, alpha 2C4, and alpha 2C2), we assessed the ability of alpha 2AR-mediated stimulation of adenylyl cyclase activity to undergo short-term agonist-promoted desensitization. To isolate alpha 2AR-Gs coupling, cells were pretreated with pertussis toxin, which ablates Gi coupling. Following agonist exposure, both alpha 2C10- and alpha 2C2-mediated stimulation of adenylyl cyclase activity underwent marked desensitization. In distinct contrast, alpha 2C4-mediated stimulation of adenylyl cyclase activity underwent no agonist-promoted desensitization. Thus, alpha 2AR-Gs coupling undergoes agonist-promoted desensitization and does so in a subtype-selective manner.

Adenylyl Cyclase Inhibitors↗

PubMed1993

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

Adenylyl Cyclases↗

PubMed1993

Pharmacology and molecular biology of adrenergic receptors.

The recent cloning of multiple adrenergic receptors has moved our understanding of these receptors from a conceptual one (Alquist, 1948) to one based on well-defined unique cellular proteins. The biochemical and pharmacological properties of these receptors can now be studied in detail by expression of a single subtype in cells normally devoid of adrenergic receptors. By site-directed mutagenesis, the relationship between the structures of these receptors and their function is now being elucidated for each adrenergic receptor subtype. These functions include the binding of catecholamines and other ligands, G protein coupling and functional regulation.

Amino Acid Sequence↗

PubMed1993

Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects.

It has long been hypothesized that a defective beta 2-adrenergic receptor (beta 2AR) may be a pathogenic factor in bronchial asthma. We examined the gene encoding the beta 2AR to assess the frequency of polymorphisms in 51 patients with moderate to severe asthma and 56 normal subjects. Nine different point mutations were found in both heterozygous and homozygous forms at nucleic acid residues 46, 79, 100, 252, 491, 523, 1053, 1098, and 1239. No mutations resulting in large deletions or frame shifts were detected. Of these nine polymorphisms, four were found to cause changes in the encoded amino acids at residues 16, 27, 34, and 164. The most frequent polymorphisms were arginine 16 to glycine (Arg16-->Gly) and glutamine 27 to glutamic acid (Gln27-->Glu). The other two polymorphisms, valine 34 to methionine, and threonine 164 to isoleucine, occurred in only four subjects. The incidence of beta 2AR homozygous polymorphisms was no greater in asthmatic patients as compared with controls (Arg16-->Gly: 53% versus 59%, Gln27-->Glu: 24% versus 29%, respectively; P = NS). Some subjects were found to have both of these polymorphisms simultaneously, but there was no difference in incidence between the two groups, with 23% of asthmatics and 28% of normal subjects being homozygous for both polymorphisms. The apparently normal subjects with both polymorphisms did not have subclinical hyperreactive airways disease as determined by methacholine challenge testing. In the asthma group, one mutation (Arg16-->Gly) identified a subset of patients with a distinct clinical profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Human alpha 2-adrenergic receptor subtype distribution: widespread and subtype-selective expression of alpha 2C10, alpha 2C4, and alpha 2C2 mRNA in multiple tissues.

At present, molecular cloning and pharmacological studies have delineated three human alpha 2-adrenergic receptor (alpha 2AR) subtypes, alpha 2C10, alpha 2C4, and alpha 2C2. Assignment of the alpha 2AR subtypes to specific functions has been limited by an unclear definition of tissue alpha 2AR expression outside of the central nervous system. It has been suggested that alpha 2C4 expression is confined to the brain, that alpha 2C2 expression is only in the liver and kidney, and that there is nearly ubiquitous expression of alpha 2C10. However, this is based on studies of a limited number of rat tissues or on studies using non-species-specific approaches. Therefore, to define alpha 2C10, alpha 2C4, and alpha 2C2 tissue expression, we used reverse transcription of total RNA isolated from 20 human tissues, followed by amplification of alpha 2AR cDNA using the polymerase chain reaction. This technique provided two advantages: high sensitivity and, with the use of subtype-specific oligonucleotide primers and probes, differentiation between the alpha 2AR subtypes. The tissues studied were aorta, vena cava, heart (epicardium and endocardium), lung, skeletal muscle, liver, pancreas (head and tail), fat (perinephric and subcutaneous), kidney (cortex and medulla), prostate, stomach, ileum, jejunum, colon, adrenal gland, and spleen. We found that the majority of these tissues expressed alpha 2C10, with the exceptions being the head of the pancreas, subcutaneous fat, colon, and spleen. In marked distinction to other studies, however, we found a prolific expression of the alpha 2C4 and alpha 2C2 subtypes. Expression of alpha 2C4 was found in all tissues with the exception of liver, fat, stomach, and colon, and a virtually ubiquitous expression of alpha 2C2 was found, with the exception of epicardium. Of all tissues studied, only colon and subcutaneous fat expressed a single alpha 2AR subtype, which was alpha 2C2. Thus, the alpha 2AR subtypes do not have a confined expression but appear to be widely distributed in humans and display subtype-specific expression in some tissues.

Base Sequence↗

PubMed1993

Lack of beta 3-adrenergic receptor mRNA expression in adipose and other metabolic tissues in the adult human.

The beta 3-adrenergic receptor (beta 3AR) has been purported to play important roles in a number of metabolic functions, suggesting that beta 3AR agonists might be useful as antidiabetic and antiobesity therapeutic agents. However, these assertions are based entirely on extensive metabolic studies with such agonists in rodents. To clarify the role that the beta 3AR might have in humans, we sought to define the tissue distribution of the beta 3AR in adult human tissue by the use of a highly specific and sensitive approach. Northern blots of selected tissues failed to reveal any beta 3AR mRNA, suggesting little or no expression. To detect minute amounts of transcripts, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) method that uses primers to amplify a region of the beta 3AR that has little homology with the closely related beta 1- and beta 2-AR genes, and we verified the specificity of this approach using plasmids containing the cloned human beta 1-, beta 2-, and beta 3AR genes. RT-PCR performed on as little as 20 ng of total RNA from 3T3-F442A cells, which expressed beta 3AR at very low levels (approximately 20 fmol/mg of protein), provided an easily detectable signal by ethidium bromide staining and Southern blotting of electrophoresed products. RT-PCR was performed on RNA obtained from 23 different human tissues, using primers for the beta 3AR, the beta 2AR, and beta-actin, which acted as a control. Whereas beta-actin and the beta 2AR were detected in virtually all tissues, RT-PCR using beta 3AR primers gave products from 13 tissues, including skeletal muscle, lung, adipose tissue, kidney, small intestine, pancreas, spleen, and adrenal gland. An end-labeled 50-nucleotide probe identical to an internal region of the expected beta 3AR product hybridized under low stringency conditions to seven of these products. However, sequencing of these products, which were somewhat smaller in molecular size than expected, did not reveal beta 3AR DNA sequence. Given the specificity and sensitivity of our approach, we conclude that the beta 3AR is not expressed to any significant degree in the adult human tissues studied, including adipose tissue and other metabolic sites.

Adipose Tissue↗

PubMed1992

Subtype-selective desensitization of alpha 2-adrenergic receptors. Different mechanisms control short and long term agonist-promoted desensitization of alpha 2C10, alpha 2C4, and alpha 2C2.

We have recently shown that the alpha 2C10 adrenergic receptor (AR) undergoes short term agonist-promoted desensitization, mediated by phosphorylation of sites in the third intracellular loop. There is significant divergence in the third loop amino acid sequences between alpha 2C10 and the other subtypes, alpha 2C4 and alpha 2C2. We therefore explored the mechanisms of alpha 2AR subtype desensitization by expressing each human subtype in Chinese hamster ovary cells and subjecting them to short and long term epinephrine exposures. After 30 min of agonist exposure, alpha 2C10 and alpha 2C2 displayed desensitization characterized by rightward shifts in the curves for epinephrine-mediated inhibition of adenylyl cyclase (EC50 = alpha 2C10, 0.24 +/- 0.02 microM increasing to 1.1 +/- 0.1 microM; alpha 2C2, 1.3 +/- 0.3 increasing to 2.6 +/- 0.3 microM). Coincident with alpha 2C10 and alpha 2C2 desensitizations were decreases in agonist high affinity binding. In contrast, alpha 2C4 underwent no functional desensitization after short term agonist exposure, nor were there any changes in agonist high affinity binding. Agonist-promoted receptor sequestration was clearly greater with alpha 2C10 (approximately 26%) and alpha 2C2 (approximately 35%) as compared to alpha 2C4 (approximately 12%), but such sequestration did not play a significant role in short term desensitization, as blockade with concanavalin A had no effect on desensitization patterns. In contrast to these findings, all alpha 2AR subtypes underwent desensitization after prolonged (24 h) agonist exposure. However, alpha 2C10 and alpha 2C2 displayed substantially more desensitization (approximately 20-fold increase in EC50) as compared to alpha 2C4 (approximately 5-fold increase). The primary mechanism of desensitization during long term agonist exposure was found to be a decrease in the amount of cellular Gi, which was equivalent in magnitude in cells expressing all three subtypes. However, in addition to a decrease in Gi, alpha 2C10 and alpha 2C2 underwent down-regulation of receptor levels during long term agonist exposure, while alpha 2C4 did not. Given that all three subtypes bind endogenous catecholamines with high affinity and inhibit adenylyl cyclase efficiently, the significance of multiple subtypes has heretofore been obscure. Our results show that alpha 2AR undergo subtype-selective desensitization to agonists and suggest that alpha 2AR subtypes may have evolved to meet differing needs for adaptive regulation.

Adenylyl Cyclase Inhibitors↗

PubMed1992

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.

Adenosine Diphosphate Ribose↗

PubMed1992

Long-term agonist exposure induces upregulation of beta 3-adrenergic receptor expression via multiple cAMP response elements.

During continuous stimulation by agonist, beta 1- and beta 2-adrenergic receptors (ARs) undergo processes that lead to decreases in receptor expression. This receptor down-regulation serves to limit the cellular cAMP response during chronic agonist exposure. In the recently described third subtype of the beta AR, denoted beta 3AR, we found four potential cAMP response elements in the 5' flanking region, suggesting that expression of this receptor might be positively regulated by agonists. These elements were cloned into the vector pA10CAT2, which contains a chloramphenicol acetyltransferase reporter gene, and transiently expressed in VERO cells. Three of these elements, TGACTCCA, TGAGGTCT, and CGAGGTCA (located 518, 622, and 1125 bases upstream of the beta 3AR coding block, respectively) were found to increase transcription of the chloramphenicol acetyltransferase gene in response to cAMP analogues and agents that increase intracellular cAMP. 3T3-F442A cells, when differentiated into the adipocyte phenotype by insulin, expressed beta 3AR, and nuclear runoff studies from such cells confirmed cAMP enhancement of beta 3AR mRNA transcription. In these cells, beta 3AR mRNA increased in response to exposure to the beta 3AR agonist isoproterenol and remained elevated during exposures of up to 24-30 hr. During prolonged exposure to agonist, no downregulation of beta 3AR expression in 3T3-F442A cells occurred. Indeed, beta 3AR expression increased during agonist exposure to approximately 165% of basal expression. In marked contrast, beta 1AR expression declined by approximately 70% in response to chronic agonist exposure. These studies reveal a subtype-specific prolonged transcriptional regulation of a beta AR gene by the end product of its signal transduction pathway. Thus, the beta 3AR undergoes a paradoxical increase in receptor expression during chronic agonist exposure.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Sites in the third intracellular loop of the alpha 2A-adrenergic receptor confer short term agonist-promoted desensitization. Evidence for a receptor kinase-mediated mechanism.

To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites.

Adenosine Diphosphate Ribose↗

PubMed1992

Electrical pacing induces adenylyl cyclase in skeletal muscle independent of the beta-adrenergic receptor.

Continuous electrical pacing (EP) at 10 Hz of the peroneal nerve innervating fast-twitch muscles of the hindlimb in adult rabbits increases skeletal muscle concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) by 3.1-fold at 10 days and increases beta-adrenergic receptor (beta-AR) density by 2.0-fold at 21 days. To determine whether beta-AR, the alpha-subunit of guanine nucleotide proteins (Gs alpha), or adenylyl cyclase is primarily responsible for pacing-induced increases in muscle cAMP, we measured adenylyl cyclase activity (ACA) in muscles that were electrically paced for 3 (n = 4), 10 (n = 8), and 21 (n = 8) days. EP resulted in a time-dependent increase in ACA that was 2.2 +/- 0.3-fold (P less than 0.005) at 21 days. EP significantly increased GTP-, 5'-guanylylimidodiphosphate-, isoproterenol-, NaF-, and forskolin-stimulated ACA, and propranolol administration to rabbits during EP did not alter pacing-induced changes in ACA. There were no changes in protein concentration, Na(+)-K(+)-adenosinetriphosphatase activity, or Gs alpha with EP. Based on these studies, we conclude that EP appears to increase cAMP through mechanisms independent of the beta-AR and through mechanisms that may involve alterations at the level of adenylyl cyclase.

Adenylyl Cyclases↗

PubMed1992

Subcellular distribution of beta 2-adrenergic receptors delineated with quantitative ultrastructural autoradiography of radioligand binding sites.

beta-Adrenergic receptors play a critical role in signal transduction in the cardiovascular system. Regulation of beta-adrenergic receptor expression depends on multiple factors, including rates of synthesis and degradation of receptor protein and trafficking of receptors between the cell surface and putative intracellular compartments. To characterize the subcellular distribution of beta-adrenergic receptors and to delineate the ultrastructure of intracellular compartments in which adrenergic receptors reside in the steady state, we studied Chinese hamster fibroblast cells transfected with the human beta 2-adrenergic receptor gene. beta 2-Adrenergic receptors of these cells were covalently labeled with the lipophilic photoactivatable antagonist [125I]iodocyanopindolol diazarine, and specific radioligand binding sites were localized at the ultrastructural level of resolution with quantitative electron microscopic autoradiography. The density of beta 2-adrenergic receptors was approximately 100-fold greater in the plasmalemma than in any other compartment. Approximately 50% of total cellular receptors were identified on the cell surface in an apparently random, nonclustered distribution and without association with clathrin-coated pits or other cell surface structural specializations. The remaining receptors were distributed among intracellular membranous compartments, including smooth vesicles, rough endoplasmic reticulum, and the Golgi apparatus, organelles presumably involved in stages of receptor synthesis, degradation, or trafficking. In the basal state, there was no association of beta 2-adrenergic receptors with coated intracellular vesicles typical of the endocytotic pathway of selected cell surface receptors that function to internalize their extracellular ligands. These results are the first to rigorously quantify the subcellular distribution of beta-adrenergic receptors and unequivocally establish the presence of a substantial pool of intracellular receptors.

Animals↗